Genetic diversity and differentiation of allopolyploid Dactylorhiza (Orchidaceae) with particular focus on the Dactylorhiza majalis ssp. traunsteineri/lapponica complex

Genetic differentiation of Dactylorhiza majalis ssp. traunsteineri from the Alps, Scandinavia, and Britain was studied and compared with other allotetraploid members of the systematically challenging genus Dactylorhiza . One-hundred and eleven populations from altogether 18 taxa were analysed for eight polymorphic plastid markers and two size-variable fragments from the nuclear internal transcribed spacer (ITS) region. In total, 60 plastid haplotypes and six ITS alleles were found among the 737 individuals analysed. No clear differentiation between populations of ssp. traunsteineri from the three regions was revealed. However, ssp. traunsteineri was genetically differentiated from Dactylorhiza baumanniana , Dactylorhiza elata , and D. majalis ssp. sphagnicola , although the majority of allotetraploid taxa remained inseparable. Judging from the degree of concerted evolution in ITS, D. majalis ssp. alpestris may be regarded as a relatively old allotetraploid, whereas ssp. baltica and ssp. purpurella may be considerably younger. Based on plastid data, the Alp region had the highest genetic diversity followed by Scandinavia and Britain. The geographic distribution of haplotypes provided support for possible refugial areas around the Alps and for several independent immigration routes into Scandinavia after the last ice age.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 52–67.     

Electrophoretic evidence for all otetraploid origin of Dactylorhiza purpurella (Orchidaceae)

Evidence from all ozyme markers suggests that the NW European Dactylorhiza purpurella (Orchidaceae) is an allotetraploid which originated from taxa closely related to the present-day D. incarnata s. I. and D. fuchsii/D. maculata . However, Dactylorhiza purpurella deviates more strongly from the allotetraploid condition than other taxa previously investigated in Dactylorhiza (i.e., D. majalis, D. traunsteineri, D. sphagnicola , and D. lapponica ), in that the characteristic incarnata alleles occur at lower frequencies than expected at two loci. It is suggested that D. purpurella arose from parents slightly different from those giving rise to the other allotetraploids, or that the tetraploid genome in D. purpurella has been modified by rare recombination events between homoeologous chromosomes, replacing segments of the incarnata chromosomes with the fuchsii/maculata genome.     

On the distinction of Dactylorhiza baltica and D. pardalina (Orchidaceae) and the systematic affinities of geographically intermediate populations

The eastern European Dactylorhiza baltica (Klinge) N. I. Orlova and the western European D. pardalina (Pugsl.) Aver. (= D. praetermissa var. junialis (Verm.) Sengh) are usually considered to have non-overlapping geographic distributions, for which reason it has rarely been realized that they are morphologically similar. They have not previously been thoroughly compared by molecular methods, and no existing flora or revision has convincingly demonstrated that they can be distinguished by morphological characters. In reality, they might be 'political' rather than natural taxa. Prompted by the recent discovery of geographically intermediate populations (in eastern Denmark), originally identified as D. baltica , we have addressed this problem by analysis of morphometric data as well as molecular data from allozyme markers, plastid haplotypes, nuclear ITS alleles and nuclear microsatellites. Dactylorhiza baltica and D. pardalina turned out to be clearly distinguished genetically, and although they are morphologically similar, a few characters were identified that distinguish with 81–85% certainty between the two taxa. Molecular and morphometric data place the geographically intermediate populations in D. pardalina . Both taxa were confirmed to be allotetraploids combining diploid genomes from the D. incarnata s.l. and D. maculata s.l. lineages, and they should therefore be recognized as infraspecic taxa under D. majalis s.l. Thus, D. baltica should be called D. majalis subsp. baltica ; D. pardalina is identical with D. praetermissa var. junialis , but the nomenclatural consequences for D. praetermissa , if treated as subspecies under D. majalis , are still unresolved.     

Plastid DNA variation in the Dactylorhiza incarnata/maculata polyploid complex and the origin of allotetraploid D. sphagnicola (Orchidaceae)

To obtain further information on the polyploid dynamics of the the Dactylorhiza incarnata/maculata polyploid complex and the origin of the allotetraploid D. sphagnicola (Orchidaceae), plastid DNA variation was studied in 400 plants from from Sweden and elsewhere in Europe and Asia Minor by means of polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) and sequencing. Allotetraploid taxa in Europe are known have evolved by multiple independent polyploidization events following hybridization between the same set of two distinct ancestral lineages. Most allotetraploids have inherited the plastid genome from parents similar to D. maculata sensu lato, which includes, e.g. the diploid D. fuchsii and the autotetraploid D. maculata sensu stricto. D. sphagnicola carries a separate plastid haplotype different from the one found in other allotetraploid taxa, which is in agreement with an independent origin from the parental lineages. Some of the remaining allotetraploids have local distributions and appear to be of postglacial origin, whereas still other allotetraploids may be of higher age, carrying plastid haplotypes that have not been encountered in present day representatives of the parental lineages. Introgression and hybridization between diploids and allotetraploids, and between different independently derived allotetraploids may further have contributed to genetic diversity at the tetraploid level. Overall, the Dactylorhiza polyploid complex illustrates how taxon diversity and genetic diversity may be replenished rapidly in a recently glaciated area.     

Amplified fragment length polymorphisms (AFLP) reveal details of polyploid evolution in Dactylorhiza (Orchidaceae)

The utility of the PCR-based AFLP technique (polymerase chain reaction; amplified fragment length polymorphisms) was explored in elucidating details of polyploid evolution in the Eurasian orchid genus Dactylorhiza. We emphasized Swedish taxa but also included some material from the British Isles and elsewhere in Europe. Three different sets of primers, amplifying different subsets of restriction fragments, independently revealed similar patterns for relationships among the Dactylorhiza samples investigated. The AFLP data support the general picture of polyploid evolution in Dactylorhiza, i.e., that allotetraploid derivatives have arisen repeatedly as a result of hybridization beween the two parental groups D. incarnata s.l. (sensu lato; diploid marsh orchids) and the D. maculata group (spotted orchids). Within the incarnata s.l. group, morphologically defined varieties were interdigitated. The D. maculata group consisted of two distinct subgroups, one containing autotetraploid D. maculata subsp. maculata and the other containing diploid D. maculata subsp. fuchsii. Allotetraploids showed a high degree of additivity for the putative parental genomes, and relationships among them were partly correlated to morphologically based entities, but also to geographic distribution. Thus, allotetraploid taxa from the British Isles clustered together, rather than with morphologically similar plants from other areas.     

Allozyme variation and genetic integrity of Dactylorhiza incarnata (Orchidaceae)

Eleven Danish populations of the diploid Dactylorhiza incarnata s.lat. have been examined for allozyme variation in DIA, PGD, PGI, PGM, SKD, TP1, and UGPP. The results reveal a deficiency of heterozygotes in several populations. Possible reasons for this deficiency are considered. The results also indicate that no genetic barrier seems to prevent gene flow between D. incarnata var. incarnata , var. dunensis , and var. ochrantha — and some indication exists that a considerable amount of gene exchange takes place between sympatric populations of var. incarnata and var. ochrantha . On the other hand, there is no sign of recent introgression between D. incarnata s.lat. and any of the sympatric tetraploid species ( D. maculata, D. majalis, D. purpurella s.lat.). It is suggested that knowledge on the various degrees of genetic integrity of morphologically recognizable entities should be elaborated and subsequently utilized for a biosystematic approach to Dactylorhiza .     

Geometric morphometrics as a tool for understanding Dactylorhiza (Orchidaceae) diversity in European Russia

Geometric morphometric techniques were employed to assess the diversity of lip shapes (305 samples from 83 populations) in flowers of European Russian Dactylorhiza (Orchidaceae: Orchidinae). We found significant agreement between the results from geometric morphometrics, classic morphometrics and the distribution of certain nuclear DNA markers. The lip shapes from Arctic Dactylorhiza samples occupied an intermediate position between D. maculata and D. fuchsii samples from Central Russia, supporting a hybrid origin of 'northern tetraploids'. Lip shapes of the taxonomically controversial allotetraploid D. baltica were found to form a distinct group, with members having definite relationships with diploid D. incarnata samples from the same localities, indicating either their local origin or introgression with D. incarnata . In addition to demonstrating the value of geometric morphometric methods in studies of plant taxonomy and hybridization, we suggest future applications designed to explore pollinator-driven directional selection, developmental constraints and fluctuating asymmetry.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 85 , 1–12.     

Dactylorhiza (Orchidaceae) in European Russia: combined molecular and morphological analysis

Four plastid and two nuclear (internal transcribed spacer [ITS] ribosomal DNA) markers were used in this study of the Dactylorhiza maculata and D. incarnata complexes (Orchidaceae: Orchidinae) to determine diversity and taxonomic distribution of haplotypes, hybridization frequencies, and maternal parentage of hybrids in 125 samples from 78 populations from European Russia and the Caucasus. A morphometric study of all populations revealed significant correspondence between morphological and plastid DNA data. Most D. maculata sensu stricto (s.s.) specimens from Russia have D. fuchsii haplotypes; this could be evidence for introgression or widespread hybridization between these species in northern Russia. Heterogeneity within populations is much higher for ITS data and is strongly correlated with latitude. Both plastid and nuclear data are significantly correlated with distribution along a south-north axis. Several haplotypes and ITS alleles uncommon in western Europe are more widely distributed in Russia, whereas some frequent haplotypes from western Europe are absent.     

Plastid DNA haplotype diversity and morphological variation in the Dactylorhiza incarnata/maculata complex (Orchidaceae) in northern Poland

To gain an overview of the variation in the Dactylorhiza incarnata/maculata complex in northern Poland, ten plastid DNA regions (seven microsatellite and three indel loci) and 23 morphometric characters were used. In total, 972 and 480 samples from 64 and 31 populations were utilized for the genetic and morphometric analyses, respectively. One hundred and forty‐one haplotypes that have not been reported previously were recognized. The continuity of morphological characters between the studied species and the impact of post‐glacial colonization on the observed complexity in the Dactylorhiza incarnata/maculata complex were concluded. It was confirmed that the allotetraploid group of D. majalis s.s. has inherited its plastid genome from D. maculata s.l., specifically from D. maculata ssp. fuchsii. In addition, some of the haplotypes found in D. majalis s.s. were distinct and evidently not present in the preserved D. maculata s.l. Although possible gene flow and introgression between two subspecies of the D. maculata s.l. group were indicated, we suggest that they should be treated as separate evolutionary units. Both the common and rare haplotypes show a similar pattern of geographical distribution for all four taxa analysed, which suggests that hybridization took place relatively recently, shortly after the retreat of the ice sheet. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 178 , 121–137.     

Nonuniform concerted evolution and chloroplast capture: heterogeneity of observed introgression patterns in three molecular data partition phylogenies of Asian Mitella (saxifragaceae)

Interspecific hybridization is one of the major factors leading to phylogenetic incongruence among loci, but the knowledge is still limited about the potential of each locus to introgress between species. By directly sequencing three DNA regions: chloroplast DNAs (matK gene and trnL-F noncoding region), the nuclear ribosomal external transcribed spacer (ETS) region, and internal transcribed spacer (ITS) regions, we construct three phylogenetic trees of Asian species of Mitella (Saxifragaceae), a genus of perennials in which natural hybrids are commonly observed. Within this genus, there is a significant topological conflict between chloroplast and nuclear phylogenies and also between the ETS and the ITS, which can be attributed to frequent hybridization within the lineage. Chloroplast DNAs show the most extensive introgression pattern, ITS regions show a moderate pattern, and the ETS region shows no evidence of introgression. Nonuniform concerted evolution best explains the difference in the introgression patterns between the ETS region and ITS regions, as the sequence heterogeneity of the ITS region within an individual genome is estimated to be twice that of an ETS in this lineage. Significant gene conversion patterns between two hybridizing taxa were observed in contiguous arrays of cloned ETS-ITS sequences, further confirming that only ITS regions have introgressed bidirectionally. The relatively slow concerted evolution in the ITS regions probably allows the coexistence of multiple alleles within a genome, whereas the strong concerted evolution in the ETS region rapidly eliminates heterogeneous alleles derived from other species, resulting in species delimitations highly concordant with those based on morphology. This finding indicates that the use of multiple molecular tools has the potential to reveal detailed organismal evolution processes involving interspecific hybridization, as an individual locus varies greatly in its potential to introgress between species.     

Systematics and conservation genetics of Dactylorhiza majalis ssp. elatior (Orchidaceae) on Gotland

A tall allotetraploid member of the Dactylorhiza incarnata/maculata complex with unspotted leaves and large pinkish flowers from the island of Gotland in the Baltic was examined for molecular variation patterns at five nuclear microsatellite loci, nuclear ITS and in plastid haplotypes. The allotetraploid was well separated from allopatric allotetraploids of similar appearance, including the western European D. majalis ssp. integrata (syn. D. praetermissa) and forms of D. majalis ssp. lapponica from mainland Sweden. It also differed from other allotetraploids distributed in the Baltic Sea region, including D. majalis ssp. baltica and D. majalis ssp. lapponica. It is here recognized as D. majalis ssp. elatior (Fr.) Hedrén & H. A. Pedersen. Dactylorhiza osiliensis Pikner, described from Saaremaa (Estonia) is regarded as a synonym. The distribution covers Gotland, Saaremaa and possibly Hiiumaa. Dactylorhiza majalis ssp. elatior may have one or several recent origins within its present distribution area, and it contains no other molecular markers than those found in the parental D. incarnata var. incarnata and D. maculata ssp. fuchsii in the same area. It appears to have weak barriers towards secondary hybridization with its parental lineages. The situation is reminiscent to that of other young allotetraploids in the D. majalis s.l. complex, suggesting that introgression may be an underestimated process explaining the accumulation of genetic diversity in evolving allopolyploid plants.     

Phylogenetic relationships and biogeographic patterns in North American members of Carex section Acrocystis (Cyperaceae) using nrDNA ITS and ETS sequence data

Carex section Acrocystis currently includes 27 taxa in North America. Recent phylogenetic studies have suggested that the North American and some but not all of the Eurasian species form a clade. Relationships and biogeographic patterns among species in this core-Acrocystis group are explored here using nuclear ribosomal (nrDNA) internal transcribed spacer region (ITS) and nrDNA external transcribed spacer region (ETS) sequence data. While maximum parsimony analysis of the ITS and ETS data provides only a moderately resolved branching structure for species relationships within the core-Acrocystis clade, maximum likelihood analysis provides a more resolved hypothesis of relationships in the section. The core-Acrocystis clade consists of a grade of Eurasian and primarily western North American species, with a well-supported clade of only eastern North American species nested within this grade. ITS and ETS types do not coalesce within many species or species complexes. Possible explanations for the non-coalescent nature of ITS and ETS copies in Acrocystis are explored, including lineage sorting, hybridization, and cryptic species.     

A secondary hybrid zone between diploid Dactylorhiza incarnata ssp. cruenta and allotetraploid D. lapponica (Orchidaceae)

Secondary hybrid zones are not uncommon in Dactylorhiza, but knowledge of ecological and evolutionary consequences of hybridization are scarce. Here, we assess interploidal gene flow and introgression in a hybrid zone between diploid Dactylorhiza incarnata ssp. cruenta (2n = 2x = 40) and its putative allotetraploid derivative D. lapponica (2n = 4x = 80). Photometric quantification of DNA content and morphology confirmed that triploids are abundant in sympatric populations in our study area. Allozyme segregation patterns in D. lapponica supported an allopolyploid origin, although unbalanced genotypes suggested rare pairings between homoeologous chromosomes. Photometric data and chromosome counts suggest backcrossing between the triploid hybrid and D. lapponica, and hence some hybrid fertility. Triploids are morphologically more similar to the tetraploids than the diploids, maybe owing to the hybrid origin of both triploids and tetraploids. The diploids and tetraploids were not more similar in the parapatric populations compared to when they occur in allopatry. This indicates that backcrossing rarely leads to introgression, or alternatively that allopatric populations are not isolated enough to prevent influx of pollen from the other species. Despite some evidence of backcrossing, our study gives few indications that widespread hybridization entails local breakdown of species boundaries. Rather, the hybrid zone may be a transient phenomenon due to intensive mowing, resulting in the opening of habitats and hence bringing the parental species into close contact.     

Nuclear ribosomal DNA sequence variation and evolution of spotted marsh-orchids (Dactylorhiza maculata group)

Sequences of both internal and external transcribed spacers of nuclear ribosomal DNA were sequenced for four species belonging to the Dactylorhiza maculata group or "spotted marsh-Orchids". These four species are D. fuchsii, D. saccifera, D. foliosa, and D. maculata. Extensive nuclear ribosomal DNA polymorphism was uncovered within the diploid D. fuchsii and the putative autotetraploid D. maculata. Within the phylogenetic trees reconstructed using parsimony and Bayesian analyses, four main lineages (A, B, C, and D) were well supported. While D. saccifera, D. maculata, and D. foliosa were confined to clades B, C, and D, respectively, D. fuchsii accessions were spread over three clades (A, B, and C). Lineage C, which included accessions of the diploid D. fuchsii and the tetraploid D. maculata, was closely related to the lineage of D. foliosa (lineage D), an endemic diploid species from Madeira. Moreover, intra-individual polymorphism was found within accessions of D. maculata, D. fuchsii, and D. saccifera. It is shown that in some instances two lineages, contributed to the observed intra-individual polymorphism (C and A in D. maculata, A and B in D. fuchsii and D. saccifera). Evolutionary scenarios leading to this extensive nuclear ribosomal DNA polymorphism are discussed in the light of results from maternally inherited chloroplast DNA markers and an autopolyploid origin of D. maculata from a D. foliosa-like ancestor is postulated.     

Dactylorhiza majalis s.l. (Orchidaceae) in acid habitats: variation patterns, taxonomy, and evolution

Morphometric and allozyme data were collected from 11 populations of Dactylorhiza majalis s.l. in the Ardennes (Belgium, France) and North Jutland (Denmark). All study populations were growing in obviously acid habitats. Genetic distances were calculated and illustrated by UPGMA dendrograms, while principal components analyses (PCAs) were used to summarize morphological variation patterns. Based on the PCAs, the probability of each character to distinguish correctly between the main groups of populations was estimated. The study populations from the Ardennes and from the Danish island of Læsø seem to agree with populations from Sweden previously studied by Hedrén—and to belong to D. majalis subsp. sphagnicola (comb. nov.). It should be noted that allozyme data suggest a polytopic origin of this taxon. The study populations from the Danish region of Thy differ morphologically from the others and exhibit much less genetic variation. They are described as a new subspecies, D. majalis subsp. calciñigiens. The evolution and biogeography of the two taxa are discussed, and brief taxonomic accounts are provided.     

Phylogenetic utility of the external transcribed spacer (ETS) of 18S-26S rDNA: congruence of ETS and ITS trees of Calycadenia (Compositae)

The 3' region of the external transcribed spacer (ETS) of 18S-26S nuclear ribosomal DNA was sequenced in 19 representatives of Calycadenia/Osmadenia and two outgroup species (Compositae) to assess its utility for phylogeny reconstruction compared to rDNA internal transcribed spacer (ITS) data. Universal primers based on plant, fungal, and animal sequences were designed to amplify the intergenic spacer (IGS) and an angiosperm primer was constructed to sequence the 3' end of the ETS in members of tribe Heliantheae. Based on these sequences, an internal ETS primer useful across Heliantheae sensu lato was designed to amplify and sequence directly the 3' ETS region in the study taxa, which were the subjects of an earlier phylogenetic investigation based on ITS sequences. Size variation in the amplified ETS region varied across taxa of Heliantheae sensu lato from approximately 350 to 700 bp, in part attributable to an approximately 200-bp tandem duplication in a common ancestor of Calycadenia/Osmadenia. Phylogenetic analysis of the 200-bp subrepeats and examination of apomorphic changes in the duplicated region demonstrate that the subrepeats in Calycadenia/Osmadenia have evolved divergently. Phylogenetic analyses of the entire amplified ETS region yielded a highly resolved strict consensus tree that is nearly identical in topology to the ITS tree, with strong bootstrap and decay support on most branches. Parsimony analyses of combined ETS and ITS data yielded a strict consensus tree that is better resolved and generally better supported than trees based on either data set analyzed separately. We calculated an approximately 1.3- to 2.4-fold higher rate of sequence evolution by nucleotide substitution in the ETS region studied than in ITS-1 + ITS-2. A similar disparity in the proportion of variable (1.3 ETS:1 ITS) and potentially informative (1.5 ETS:1 ITS) sites was observed for the ingroup. Levels of homoplasy are similar in the ETS and ITS data. We conclude that the ETS holds great promise for augmenting ITS data for phylogenetic studies of young lineages.     

Sequence analyses of the ITS regions and the matK gene for determining phylogenetic relationships of Diospyros kaki (persimmon) with other wild Diospyros (Ebenaceae) species

To elucidate the relationships among Diospyros kaki and species closely related in previous studies, the nuclear ribosomal internal transcribed spacer (ITS) DNA sequence and the chloroplast matK gene were sequenced and compared with those of nine Diospyros species from Thailand, four species from temperate regions, and one species of southern Africa, D. lycioides. Maximum parsimony, maximum likelihood, and neighbor joining analyses of the matK and ITS data sets revealed that D. kaki is closely related to two diploid species, D. oleifera and D. glandulosa. D. kaki, D. glandulosa, and D. oleifera were placed differently in the trees obtained from ITS and matK data sets, suggesting that hybridization and/or introgression may have occurred during the development of these species. D. kaki was not found to be closely related to D. ehretioides, a diploid species from Thailand. These results differed from a prior analysis of this genus performed with chloroplast DNA (cpDNA) restriction site mutations in 3.2- and 2.1-kb amplified sequences. The results supported Ng’s hypothesis that D. glandulosa and D. kaki may share a common ancestor. D. oleifera was also closely associated with D. kaki.     

Patterns of polyploid evolution in Greek marsh orchids (Dactylorhiza; Orchidaceae) as revealed by allozymes, AFLPs, and plastid DNA data

Polyploidy is common in higher plants, and speciation in polyploid complexes is usually the result of reticulate evolution. We examined variation in nuclear AFLP fingerprints, nuclear isozymes, and hypervariable plastid DNA loci to describe speciation patterns and species relationships in the Dactylorhiza incarnata/maculata polyploid complex (marsh orchids; Orchidaceae) in Greece. Several endemic taxa with restricted distribution have been described from this area, and to propose meaningful conservation priorities, detailed relationships need to be known. We identified four independently derived allopolyploid lineages, which is a pattern poorly correlated with prevailing taxonomy. Three lineages were composed of populations restricted to small areas and may be of recent origins from extant parental lineages. One lineage with wide distribution in northern Greece was characterized by several unique plastid haplotypes that were phylogenetically related and evidently older. The D. incarnata/maculata polyploid complex in Greece has high levels of genetic diversity at the polyploid level. This diversity has accumulated over a long time and may include genetic variants originating from now extinct parental populations. Our data also indicate that the Balkans may have constituted an important refuge from which northern European Dactylorhiza were recruited after the Weichselian ice age.